The cell-specific concentrations and interactions of the estrogen receptor (ER) isoforms and ER coregulators (activators and repressors) in target cells are now believed to explain the tissue specific actions of selective estrogen receptor (ER) modulators (SERMs). Estrogens (E) and SERMs modulate the structure of the ligand binding domain (LBD) of the ER which, in turn, regulates the interactions of the nuclear coregulators with the ER and determines the actions of the ER on gene expression and subsequent tissue responses. Studies supported by this grant over the past 3 1/2 years have yielded information on the actions of ERalpha or ERbeta (or combinations of the two) on gene expression in osteoblasts (OB) and their effects on OB function. Using these results and our new OB cell lines, derived from human and murine knockout skeletal tissues, we propose the following Aims for this project. Aim 1: Examine the synergism/antagonism of the ERalpha and ERbeta isoforms on gene transcription of selected endogenous genes [IL-6, alkaline phosphatase (AP), progesterone receptor (PR), TGFbeta inducible early gene (TIEG), and veriscan (Ver)] in novel human OB (U2OS) cell lines. Aim 2: Determine the effects of reduced/absent SRC-1 and SRC-2 co-activator levels on the ERalpha and ERbeta-regulated transcription by E and SERMs of the same endogenous genes (as in Aim 1) in human OB: a) Transfect Dharmacon siRNAs with oligofectamine in our newly created human OB (U2OS) cells containing tetracycline regulated expressions of ERalpha, or ERbeta, or both ERalpha/beta, and b) perform the same studies as in Aim 2-a using immortalized mouse OB from wild-type and SRC-1 and SRC-2 KO mice. Aim 3: Determine the molecular interactions of the ER (alpha and beta) and its co-regulators (SRC-1, -2, -3, p300, CBP, CARM1, N-CoR, RIP 140, and REA) using GST pull-down studies with GST-ERalpha- and GST-ERbeta-LBD fusion proteins when the LBD is bound with E or SERMs. Identify bound co-regulators by western blotting. Aim 4: Determine the complete profile of the OB-derived co-regulators bound to the GST-ERalpha and GST-ERbeta LBD fusion proteins, when bound with E or the SERMs, using 1D-PAGE as well as 3D nano-high performance liquid chromatography coupled directly with tandem mass spectrometry and use the data to search genomic and protein databases for characterization and identification.